Population studies have shown an inverse relationship between plasma high-density lipoprotein (HDL) levels and risk for cardiovascular disease, implying that factors associated with HDL metabolism are atheroprotective. One of these factors is a cell membrane ATP-binding cassette (ABC) transporter called ABCA1, which mediates the transport of excess cholesterol from cells to HDL apolipoproteins. Although ABCA1 expression is highly regulated at the level of transcription, ABCA1 protein expression and activity are also modulated by poorly understood post-translational processes. The interaction of apolipoproteins with cells has at least three distinct effects on the ABCA1 pathway, including removal of ABCA1-transported cholesterol and phospholipids, stabilization of ABCA1 protein, and activation of a tyrosine kinase signaling pathway required for ABCA1 function. The apolipoprotein and ABCA1 determinants responsible for these effects are unknown. These and other studies have implicated multiple cellular factors as being involved in stabilizing ABCA1 protein and regulating its activity. We propose to use functional assays and protein discovery strategies to characterize processes involved in modulating ABCA1 protein levels and function. We will characterize the effects of apoA-I and apolipoprotein-mimetic peptides on ABCA1 expression and activity. These studies will use a variety of synthetic peptides and ABCA1 mutants to establish the apolipoprotein and ABCA1 properties responsible for this modulation, to characterize changes in ABCA1 conformation induced by apolipoproteins, and to identify sites in ABCA1 that interact with apolipoproteins. We will test for the ability of ABCA1-stabilizing peptides to protect against atherosclerosis in mouse models. We will also identify and characterize cellular factors that modulate ABCA1 activity and protein levels using in vitro activity assays, mass spectrometric analysis of proteins, and insertional mutagenic gene discovery. These studies will suggest possible sites of impairment of the ABCA1 pathway and uncover potential therapeutic targets for treating cardiovascular disease.